Dysferlin: Approaches to Domain Analysis and Gene Therapy

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Membrane repair is a fundamental cell survival process in large, irreplaceable, or frequently injured cell types such as muscle cells. We have reported that the skeletal muscle protein dysferlin is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and that dysferlin accelerates muscle membrane repair via a calcium-triggered mechanism that invokes binding to annexins and the formation of intracellar vesicular aggregates. It thus seems likely that MM and LGMD2B are a consequence of aberrant skeletal muscle membrane repair. We now propose to define domains of dysferlin that are critical for this repair process and determine whether truncated forms of "mini-dysferlin" can rescue the MM/LGMD2B phenotype. Our study has five Specific Aims: (1) Generate and characterize transgenic mice that over- express full-length and truncated dysferlin. Hypothesis: expression of supra-normal levels of full-length or truncated dysferlin can rescue the dystrophic phenotype in a dysferlin-deficient model. (2) Characterize the biochemical properties of truncated dysferlin. Hypothesis: A biochemical analysis of full-length and truncated dysferlin proteins will identify domains retaining functional properties of the full-length protein. (3) Characterize membrane repair by mini-dysferlin proteins. Hypothesis: mini-dysferlin proteins retaining functional domains will augment membrane repair in dysferlin-deficient muscle cells. (4) Intravenously deliver candidate AAV8-packaged mini-dysferlin genes. Hypothesis: systemic delivery of mini-dysferlin genes will give rise to dissemination of these genes in muscle and enhanced membrane repair. (5) Analyze zebrafish for the presence of dysferlin and for phenotypes when dysferlin homologues are down-regulated. Hypothesis: Dysferlin-deficiency will produce a myopathic phenotype in zebrafish and it will be possible to analyze the impact of replacement of partial or full forms of dysferlin in the zebrafish. Methods: We will use a variety of techniques to define functional domains of dysferlin and will then analyze the rescue of the dysferlin-deficient phenotypes by full length and truncated dysferlin delivered to muscle in vivo via transgenic mice and systemically administered AAV8. Significance: This study willl (1) enhance our understanding of the molecular biology of sarcolemmal membrane repair in skeletal muscle; (2) provide insight into the functional domains of dysferlin; (3) determine the feasibility of developing mini-dysferlins for rescue of muscle phenotypes resulting from dysferlin deficiency; and (4) develop pilot data on the efficacy of systemically administered viral gene therapy for MM and LGMD2B.